Methods for detecting the presence of biological substances and chemical compounds in samples has been an area of continuous development in the field of analytical chemistry and biochemistry. Several types of assay currently exist for detecting the presence of target species or analytes in samples. Such methods include the radioimmunoassay (RIA) and the enzyme-linked immunosorbent assay (ELISA). Kuby, J., Immunology, W. H. Freeman and Company, New York, N.Y., pp. 147-150 (1991). Although ELISA and other immunosorbent assays are simple and widely used methods, they have several disadvantages. For example, labeled antibodies are often expensive, especially for assays requiring radioactive labels. Additionally, radioactive labels require special handling as radioactive materials. Furthermore, the labeling of a compound, may alter the binding affinity of antibody to analyte.
The direct detection of the binding of proteins and ligands has also been investigated using surface plasmon reflectometry (SPR). Schmitt, F.-J.; Haussling, L.; Ringsdorf, H.; Knoll, W., Thin Solid Films, 210/211, pp. 815 (1992); Haussling, L.; Ringsdorf, H. Langmuir, 7, pp. 1837 (1991). SPR is sensitive to changes in the index of refraction of a fluid near a thin metal surface that has been excited by evanescent electromagnetic waves. The binding of proteins to ligands may be detected by examining an increase in the resonance angle or intensity of signal. Typical angular resolution using this method is 0.005° allowing detection of sub-angstrom changes in adsorbed film thickness with SPR. However, care must be taken to ensure that the change in resonance angle is due to binding and not just a change in the bulk solution index of refraction. A thermally stable environment is required due to the dependence of the resonance angle on the index of refraction of the fluid. An increase in temperature from 25° C. to 26° C. in water amounts to a change in the index of refraction by 0.0001. This increase would result in the change in resonance angle of approximately 0.015° or roughly 0.2 nm in the observed height of a protein layer. This temperature stability requirement makes SPR unsuitable for most field applications. In addition, non-specific adsorption of molecules onto or near the sensor surface can lead to false changes in signal.
A method based on a porous silicon support that permits optical detection of the binding of specific proteins to ligands has also been disclosed. Lin, V.; Motesharei, K.; Dancil, K. S.; Sailor, M. J.; Ghadiri, M. R., Science, 278, pp. 840 (1997); Dancil, K. S.; Greiner, D. P.; Sailor M. J., J. Am. Chem. Soc., 121, pp. 7925 (1999). The porous areas are typically 1 to 5 μm deep and a few square micrometers to millimeters in area. Binding of streptavidin to biotinylated surfaces was initially found to reduce the index of refraction of the porous support, however this was later correctly attributed to surface oxidation.
The use of polymerized multilayer assemblies for the detection of receptor-ligand interactions has also been disclosed. Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D., Science, 261, pp. 585 (1993); Pan, J. J.; Charych, D., Langmuir, 13, pp. 1365 (1997). Polydiacetylene multilayer films deposited by Langmuir-Blodgett technique change color from blue to red due to a conformational change in the polymer backbone. The response may be controlled and used for protein detection by attaching ligands to the multilayer.
Recently, several assay devices that utilize liquid crystals have been disclosed. For example, a liquid crystal assay device using mixed self-assembled monolayers (SAMs) containing octanethiol and biotin supported on an anisotropic gold film obliquely deposited on glass has recently been reported. Gupta, V. K.; Skaife, J. J.; Dubrovsky, T. B., Abbott N. L. Science, 279, pp. 2077-2079 (1998). In addition, PCT publication WO 99/63329 discloses assay devices using SAMs attached to a substrate and liquid crystal layer which is anchored by the SAM. PCT publication WO 01/61357 discloses a method for detecting pathogens using a liquid crystal and a substrate with a detection region that includes depressions which may be grooves. A blocking material such as bovine serum albumin may be used in conjunction with a binding agent such as an immunoglobulin to bind a pathogenic agent such as a virus or bacteria. A liquid crystal is used to detect whether the pathogenic agent is present in a sample.
Although many of the conventional assay methods described above work very well to detect the presence of target species, these methods are often expensive and usually require instrumentation and highly trained individuals. This makes such methods difficult to use in the field. Thus, a need exists for assay devices and systems which are easier to use and which allow for evaluation of samples in remote locations.
A need remains for convenient methods that may be used to detect the presence of analytes in a sample. A need also remains for a method that may be used to image phenomena that occur on a surface of a substrate.